Approximately 25% of cytoplasmic tRNAs in eukaryotic organisms experience U34 modification at C5, and the modification is thought to be unique to the eukaryotic organisms. Lack of tRNA U34 modification has a disproportionately detrimental effect on translation of several important proteins, due to the encoding genes of which use biased codons that require the modified U34 in tRNAs for efficient translation. Genetic studies indicated that the Elongator complex is responsible for the early stages of the modification. The Elongator complex was initially isolated as part of hyperphosphorylated RNA polymerase II holoenzyme. It has been shown to be involved in a variety of different cellular activities. Defects in the Elongator complex have been linked to several neurological diseases, such as familial dysautonomia (FD), rolandic epilepsy (RE), and amyotrophic lateral sclerosis (ALS). Although the Elongator complex also has been shown to possess histone acetylation activity, accumulating evidence in the literature indicates that tRNA U34 modification at C5 plays a major role in its cellular functions. The Elongator complex is a large macromolecular complex consisting of six subunits of the Elongator proteins, Elp1-6. The third subunit Elp3 is generally regarded as the catalytic subunit, but the mechanism how the Elongator complex carries out tRNA U34 modification at C5 is unknown. Through bioinformatic analysis, we found that the catalytic subunit Elp3, but not other subunits of the eukaryotic Elongator complex, is present in most archaea, a small number of bacteria, and two viruses. Based on this and other bioinformatic analyses, we propose that, unlike tRNA U34 modification in eukaryotic organisms that requires all six subunits of the Elongator complex, Elp3 alone is sufficient for the same modification reaction in archaea and bacteria. Therefore, archaeal or bacterial Elp3 provides us a simplified platform for the study of the mechanism of tRNA U34 modification at C5. In this application, we will utilize expertise from four laboratories to carry out research with three specific aims. First, we will provide the evidence that Elp3 is involved in tRNA U34 modification at C5 in archaea and bacteria by characterizing the modified U34 in tRNAs isolated from these species. Second, we will perform in vitro reconstitution of tRNA U34 modification at C5 using the recombinant archaeal Elp3 and the Elongator complex isolated from yeast cells. Third, we will carry out structural studies of Elp3 alone as well as in complex with tRNA. The long-term goal of this project is to elucidate the structure and function of the eukaryotic Elongator complex, which, while maintaining the main and evolutionarily ancient function of tRNA U34 modification at C5, may have acquired additional biochemical functions over the course of evolution by recruiting Elp1-2 and Elp4-6.